SUMMARY Cryptosporidium is an AIDS-OI pathogen for which effective treatments are yet unavailable. It is also one of the top diarrheal-causing pathogens afflicting children in developing countries, causing significantly increased mortality and stunted growth. Humans acquire cryptosporidial infection by ingesting oocysts, from which sporozoites are released to infect gastrointestinal epithelial cells. The invasion of cryptosporidial sporozoites is a first step to initialize the infection, but the key molecules in host cells interacting with the parasite during the invasion remain unknown. The long-term goal of this project is to elucidate the molecular mechanism regulating the cryptosporidial invasion. Towards the goal, we initiated a UV irradiation-based host cell mutagenesis study, in which we produced 43 host cell mutants derived from the parent wild-type (WT) HCT-8 cells and identified one of the mutant (i.e., A05 mutant) was significantly defective in the parasite attachment and invasion. In comparison to WT cells, A05 cells are much less stretched out on the surface of culture plates, and cell-to-cell connections are generally loose, leaving apparent gaps between neighboring cells. Our central hypothesis is that a host cell membrane protein (or a protein complex) is required for the attachment and invasion by cryptosporidial sporozoites. The availability of A05 mutant makes it possible to identify mutated and/or significantly regulated genes that can be prioritized for individually validation of their role in the parasite attachment and invasion. This R21 proposal represents an early stage of forward genetic studies towards our long-term goal by achieving the following two specific aims. In aim 1, we will identify mutated and significantly regulated genes by transcriptomic and proteomic analyses. RNA-seq-based transcriptome analysis will be utilized to identity protein-coding genes in the A05 mutant that are mutated and/or significantly regulated. Proteomic technology focus on identifying membrane proteins that are mutated and/or significantly regulated. Identified genes/proteins will be prioritized for functional validation. In aim 2, we will validation of the function of candidate genes in the parasite attachment and invasion. Based on the prioritized candidate genes in aim 1, their role in the attachment and invasion by sporozoites will be evaluated by antibody blocking assay, and by generating genetically modified cell lines from the WT HCT-8 cells for testing their susceptibility to the attachment and invasion. This proposal focuses on identifying gene mutations associated with the resistance to the attachment and invasion by Cryptosporidium sporozoites in A05 mutant for subsequent delineation of the molecular mechanism regulating the parasite attachment and invasion.